Hydrophilic interaction chromatography coupled to ultraviolet photodissociation affords identification, localization, and relative quantitation of glycans on intact glycoproteins
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo Sapiens (human)
SUBMITTER: Jennifer Brodbelt
PROVIDER: PXD050098 | JPOST Repository | Sat Feb 24 00:00:00 GMT 2024
REPOSITORIES: jPOST
Similar Datasets
Project description:ER+ve and ER-ve breast cancers tend to show different patterns of metastasis, with ER+ve tumours having a propensity to metastasize to the bone while ER-ve tumours tend to induce visceral metastasis. Glycans can play an important role in metastasis through their interactions with glycan binding proteins. Moreover we, and others have also shown that expression of particular tumor-associated glycoforms of the mucin MUC1, allows it to interact with lectins of the immune system. We now have data showing differences in the level of expression of some glycosyltransferases in ER+ve and ER-ve of breast cancer suggesting the expression of different O-linked glycoforms.
2011-09-26 | GSE32394 | GEO
Project description:We report the use of hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) to profile intact glycoform distributions of high mannose-type N-glycosylated proteins, using an industrially produced fungal lipase for the food industry as an example. We compared these results with conventional reversed phase LC-MS (RPLC-MS) and sodium dodecyl sulfate–polyacrylamide gel-electrophoresis (SDS-PAGE). HILIC appeared superior in resolving lipase heterogeneity, facilitating mass assignment of N-glycoforms and sequence variants. Fractions representing the four main HILIC elution bands for lipase were subjected to SDS-PAGE confirming that HILIC retention increased with the number of glycosylation sites (0-3) occupied. Additional bottom-up proteomic analysis of the fractions enabled the identification of the most abundant glycosylation sites. Compared to RPLC-MS, HILIC-MS provided a stronger reduction of the sample complexity delivered to the mass spectrometer, facilitating the assignment of the masses of glycoforms and sequence variants as well as increasing the number of glycoforms detected (69 more proteoforms, 177% increase). The HILIC-MS method required relatively short analysis time (< 30 min), in which over 100 glycoforms were distinguished. We suggest that HILIC-MS can be used to characterize bioengineering processes aimed at steering protein glycoform expression as well as to check the consistency of product batches.
2020-05-04 | PXD016215 | Pride
Project description:ER+ve and ER-ve breast cancers tend to show different patterns of metastasis, with ER+ve tumours having a propensity to metastasize to the bone while ER-ve tumours tend to induce visceral metastasis. Glycans can play an important role in metastasis through their interactions with glycan binding proteins. Moreover we, and others have also shown that expression of particular tumor-associated glycoforms of the mucin MUC1, allows it to interact with lectins of the immune system. We now have data showing differences in the level of expression of some glycosyltransferases in ER+ve and ER-ve of breast cancer suggesting the expression of different O-linked glycoforms. RNA will be isolated from ER+ve and ER-ve primary breast cancers supplied by the Guy’s and St Thomas’ Research Breast Tissue Bank. Tumor tissue will be macro-dissected from thick cryostat sections by visual comparison with a representative H&E section. We will verify the level of expression of ER and document the expression of ST3Gal-I and ST6GalNAc-II by RT-qPCR. We have already isolated RNA from a number of tumours and shown we can obtain RNA of good quality with a RNA integrity number, as measured with an Agilent Bioanalyser, of >4.5 Since there is likely to be variability among primary tumors, ideally we analyzed of 10 ER negative and 9 ER positive tumours.
2011-09-25 | E-GEOD-32394 | biostudies-arrayexpress
Project description:Proteomics has exposed a plethora of post-translational modifications, but demonstrating functional relevance requires new approaches. Top-down proteomics can characterize co-occurring modifications in terms of localization, abundance and hierarchy. Here, we present a top-down MS analysis workflow for the discovery and quantification of proteoforms. Confident fragment assignment allows for localization of modification sites and quantification of all proteoforms, including positional isomers, as validated by investigating synthetic isoforms of ubiquitin and hyper-phosphorylated Bora.
2021-03-18 | PXD012084 | Pride
Project description:Alterations of protein abundance and post-translational modifications in patients with Alzheimer’s disease (AD), such as glycosylation, phosphorylation, and ubiquitination, and their roles in disease progression and treatment outcome are areas of intense study. Little is known, however, about the overall N-glycosylation of proteins in human brains, and those from Alzheimer’s patients, particularly in regard to large-scale intact N-linked glycoproteomics analysis. To elucidate the glycoproteome landscape, we developed an approach based on multi-lectin affinity enrichment, hydrophilic interaction chromatography (HILIC), and LC-MS-based glycoproteomics analysis. We analyzed 10 normal, 10 asymptomatic, and 10 symptomatic AD brains, in which we detected >300 glycoproteins and >1,900 glycoforms across the samples. The majority of glycoproteins have N-glycans that are high-mannosidic and complex chains that are fucosylated and bisected. The Man5 N-glycan was found to occur most frequently at >20% of the total glycoforms. Unlike the glycoproteomes of other tissues, sialylation is a minor feature of the brain glycoproteome, occurring at <9% of N-glycans . We observed changes in the number of antennae, frequency of fucosylation, bisection, and other monosaccharides at individual glycosylation sites among normal, asymptomatic, and symptomatic AD samples. Further analysis revealed glycosylation differences in subcellular compartments. We did not observe a statistical difference between male and female patients. These results represent the first glycoproteomics landscape of brains from multiple AD individuals, which will facilitate a deeper understanding of AD and possible disease treatment options.
2022-10-31 | PXD032219 | Pride
Project description:Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes an extensively glycosylated surface spike (S) protein to mediate host cell entry and the S protein glycosylation plays key roles in altering viral binding/function and infectivity. However, the molecular structures and glycan heterogeneity of the new O-glycans found on the S protein regional-binding domain (S-RBD) remain cryptic because of the challenges in intact glycoform analysis by conventional bottom-up glycoproteomic approaches. Here, we report the complete structural elucidation of intact O-glycan proteoforms through a hybrid native and denaturing top-down mass spectrometry (MS) approach employing both trapped ion mobility spectrometry (TIMS) quadrupole time-of-flight and ultrahigh-resolution Fourier transform ion cyclotron resonance (FTICR)-MS. Native top-down TIMS-MS/MS separates the protein conformers of the S-RBD to reveal their gas-phase structural heterogeneity, and top-down FTICR-MS/MS provides in-depth glycoform analysis for unambiguous identification of the glycan structures and their glycosites. A total of eight O-glycoforms and their relative molecular abundance are structurally elucidated for the first time. These findings demonstrate that this hybrid top-down MS approach can provide a high-resolution proteoform-resolved mapping of diverse O-glycoforms of the S glycoprotein, which lays a strong molecular foundation to uncover the functional roles of their O-glycans. This proteoform-resolved approach can be applied to reveal the structural O-glycoform heterogeneity of emergent SARS-CoV-2 S-RBD variants, as well as other O-glycoproteins in general.
2021-07-27 | MSV000087891 | MassIVE
Project description:The presence of lunasin was investigated in two soybean products, raw soy seeds and a commercial soybean beverage powder. Lunasin from the commercial soybean derivative was purified by reverse phase high pressure liquid chromatography and bystrong anion exchange solid phase extraction. Lunasin was further characterized by Top-down mass spectrometry (MS). The top-down characterization of lunasin unraveled a wide range of post-translationally modified proteoforms.
2016-05-24 | PXD003064 | Pride
Project description:Human plasma fibronectin is an adhesive protein that plays a crucial role in wound healing. Many studies had indicated that glycans may mediate the expression and functions of fibronectin, yet a comprehensive understanding of its glycosylation is still missing. Here, we performed a comprehensive N- and O-glycosylation mapping of human plasma fibronectin, and quantified the occurrence of each glycoform in a site-specific manner. Intact N-glycopeptides were enriched by zwitterionic hydrophilic interaction chromatography, and N-glycosites sites were localized by the 18O-labeling method. O-glycopeptide enrichment and O-glycosite identification were achieved by an enzyme-assisted site-specific extraction method. An RP–LC–MS/MS system functionalized with Collision-Induced Dissociation and stepped normalized collision energy (sNCE)-HCD tandem mass was applied to analyze the glycoforms of fibronectin. A total of 6 N-glycosites and 53 O-glycosites were identified, which were occupied by 3842 N-glycoforms and 16 O-glycoforms, respectively. Furthermore, 81.4% of N-glycans were either fucosylated, sialylated, or with both modifications77.31% of N-glycans were sialylated, while O-glycosylation was dominated by the sialyl-T antigen. These site-specific glycosylation patterns on human fibronectin can facilitate functional analyses of fibronectin and therapeutics development.
2022-02-17 | PXD025886 | Pride
Project description:The health state of an individual is closely linked to the glycosylation patterns of their blood plasma proteins. However, obtaining this information requires cost- and time-efficient analytical methods. We put forward infrared spectroscopy, which allows label-free analysis of protein glycosylation, but so far has only been applied to analyses of individual proteins. Although spectral information does not directly provide the molecular structure of the glycans, it is sensitive to changes therein and covers all types of glycosidic linkages. Combining single-step ion exchange chromatography with infrared spectroscopy, we developed a workflow that enables separation and analysis of major protein classes in blood plasma. Our results demonstrate that infrared spectroscopy can identify different patterns and global levels of glycosylation of intact plasma proteins. To showcase the strengths and limitations of the proposed approach, we compare the glycoforms of human and bovine alpha-1-acid glycoproteins, which exhibit highly variable global levels of glycosylation. To further independently evaluate our conclusions, the glycan moieties of human alpha-1-acid glycoprotein were further analyzed using established glycomics workflow. Importantly, the chromatographic separation of blood plasma improves the detection of aberrant glycoforms of a given protein, as compared to infrared spectroscopy of bulk plasma. The presented approach allows time-efficient comparison of glycosylation patterns of multiple plasma proteins, opening new avenues for biomedical probing.
2024-02-26 | PXD046796 | Pride
Project description:Top-down analysis of intact proteins by mass spectrometry provides an ideal platform for comprehensive proteoform characterization, in particular, for the identification and localization of post-translational modifications (PTM) co-occurring on a protein. One of the main bottlenecks in top-down proteomics is insufficient protein sequence coverage caused by incomplete protein fragmentation. Based on previous work on peptides, increasing sequence coverage and PTM localization by combining sequential ETD and HCD fragmentation in a single fragmentation event, we hypothesized that protein sequence coverage and phospho-proteoform characterization could be equally improved by this new dual fragmentation method termed EThcD, recently been made available on the Orbitrap Fusion. Here, we systematically benchmark the performance of several (hybrid) fragmentation methods for intact protein analysis on an Orbitrap Fusion, using as a model system a 17.5 kDa N-terminal fragment of the mitotic regulator Bora. During cell division Bora becomes multiply phosphorylated by a variety of cell cycle kinases, including Aurora A and Plk1, albeit at distinctive sites. Here, we monitor the phosphorylation of Bora by Aurora A and Plk1, analyzing the generated distinctive phospho-proteoforms by top-down fragmentation. We show that EThcD and ETciD on a Fusion are feasible and capable of providing richer fragmentation spectra compared to HCD or ETD alone, increasing protein sequence coverage, and thereby facilitating phosphosite localization and the determination of kinase specific phosphorylation sites in these phospho-proteoforms.
2015-07-16 | PXD001845 | Pride